The long-term goal of this project is to determine the molecular basis of herpes simplex virus (HSV) invasion of human cells and spread of the virus to neuronal tissues. HSV infection is a major health problem worldwide infecting 60-90% of the world's population, many of which are plagued by initial and recurrent lesions for their entire lives. Adding to that is the morbidity and the risk of blindness from ocular lesions and even lethality of HSV disease in neonates and in increasing number of immunocompromised individuals. The 3-O-sulfated heparan sulfate (3-OS HS) serves as a specific entry receptor for HSV type-1 by offering binding sites for envelope glycoprotein D (gD1). Some recent data suggests that 3-OS HS can mediate all forms of primary infection, from HSV-1 entry into cells to cell-to-cell spread of the virus by mediating cell fusion. The cell-to-cell fusion as well as the fusion of the viral envelope with host cell membrane appear to require gD/3-OS HS complex in concert with three additional viral glycoproteins, gB, gH and gL. It is not clear how the receptor binding triggers the sequence of events that result in fusion. Extensive studies involving gD1 (wild-type and mutant forms), gD2, and 3-OS HS variants will be performed to develop a better understanding of the structural and biochemical requirements for a productive interaction between gD and 3-OS HS. Multiple assays that include in vitro and in vivo binding of 3-OS HS with gD, cell fusion, and viral entry will be used. Further, to determine the significance of 3-OS HS in HSV-1 entry, studies will be performed in some natural target cells to examine expression and the potential contribution of 3-OS HS in tissue tropism. These studies will be aided by the use of primary cell cultures, protein expression assays, novel peptides generated by phage display screening, and siRNA (small-interfering RNA) technology. The reagents generated would also be useful for evaluating other biological functions of 3-OS HS as well.